Estrogenic substances are commonly used in methods of Hormone Replacement Therapy (HRT) and methods of female contraception. These estrogenic substances can be divided in natural estrogens and synthetic estrogens. Examples of natural estrogens that have found pharmaceutical application include estradiol, estrone, estriol and conjugated equine estrogens. Examples of synthetic estrogens, which offer the advantage of high oral bioavailability include ethinyl estradiol and mestranol.
Recently, estetrol has been found effective as an estrogenic substance for use in HRT, disclosure of which is given in the Applicant's co-pending application WO 02/094276. Estetrol is a biogenic estrogen that is endogeneously produced by the fetal liver during human pregnancy. Other important applications of estetrol are in the fields of contraception, therapy of auto-immune diseases, prevention and therapy of breast and colon tumors, enhancement of libido, skin care, and wound healing as described in the Applicant's co-pending applications WO 02/094276, WO 02/094279, WO 02/094278, WO 02/094275, EP 2077272.9, EP 2077273.7, WO 03/041718, WO 03/018026, EP 2077812.2, and EP 2077322.2.
The synthesis of estetrol and derivatives thereof on a laboratory scale basis is known in the art: Fishman J., Guzik H., J. Org. Chem. 33, 3133-3135 (1968); Nambara T. et al., Steroids 27, 111-121 (1976); or Suzuki E. et al., Steroids 60, 277-284 (1995).
Fishman J., Guzik H., J. Org. Chem. 33, 3133-3135 (1968) discloses a successful synthesis of estetrol from an estrone derivative (compound (III); cf. for a synthesis of compound (III) Cantrall, E. W., Littell, R., Bernstein, S. J. Org. Chem 29, 214-217 (1964)). In a first step, the carbonyl group at C17 of compound (III) was reduced with LiAlH4 to estra-1,3,5(10),15-tetraene-3,17-diol (compound VIa) that was isolated as the diacetate (compound VIb). Compound VIb was subjected to cis-hydroxylation of the double bond of ring D by using OsO4 which resulted into the formation of estra-1,3,5(10)-triene-3,15α,16α,17β-tetraol-3,17-diacetate (compound Ib) that under heating with K2CO3 in methanol produces estetrol (Scheme 1).

The overall yield of this three step process is, starting from estrone derivative III, only about 7%. It is worth noting that the protected derivative 17,17-ethylenedioxyestra-1,3,5(10),15-tetraene-3-ol-3-acetate (compound IV) could be cis-hydroxylated to its 15α,16α-diol derivative (compound Va), but that thereafter the dioxolane group could not be removed (p-toluene sulfonic acid in acetone at room temperature) or that the hydrolysis (aqueous sulfuric acid in warm dioxane) of the dioxolane group resulted in a mixture containing a multitude of products (Scheme 2).

Nambara T. et al., Steroids 27, 111-121 (1976) discloses another synthesis of estetrol wherein estrone is the starting material. The carbonyl group of estrone is first protected by treatment with ethylene glycol and pyridine hydrochloride followed by acetylation of the hydroxy group at C3. The next sequence of steps involved a bromination/base catalyzed dehydrobromination resulting into the formation of 17,17-ethylenedioxyestra-1,3,5(10),15-tetraene-3-ol (compound IVa). This compound IVa was subsequently acetylated which produced 17,17-ethylenedioxyestra-1,3,5(10),15-tetraene-3-ol-3-acetate (compound IVb). In a next step, the dioxolane group of compound IVb was hydrolysed by using p-toluene sulfonic acid to compound Vb, followed subsequently by reduction of the carbonyl group at C17 (compound Vc) and oxidation of the double bond of ring D thereby forming estra-1,3,5(10)-triene-3,15α,16α,17β-tetraol-3,17-diacetate (compound VIb). See Scheme 3.
Suzuki E. et al., Steroids 60, 277-284 (1995) also discloses the synthesis of estetrol by using compound Vb of Nambara T. et al. as starting material. The carbonyl group at C17 of this compound was first reduced followed by acetylation yielding estra-1,3,5(10),15-tetraene-3,17-diol-3,17-diacetate (compound 2b). The latter was subjected to oxidation with OsO4 which provided estra-1,3,5(10)-triene-3,15α,16α,17β-tetraol-3,17-diacetate (compound 3b) in 46% yield.

According to the Nambara T. et al. and Suzuki E. et al., the synthesis of estetrol can be performed with a yield of approximately 8%, starting from estrone.
Poirier D., et al., Tetrahedron 47, 7751-7766 (1991) discloses the following compounds which were prepared according to methods that have been used to prepare similar compounds:

Dionne, P. et al., Steriods 62, 674-681 (1997) discloses the compound shown above wherein R is either methyl or t-butyldimethylsilyl.
Magnus, P. et al., J. Am. Chem. Soc. 120, 12486-12499 (1998) discloses that the main methods for the synthesis of α,β-unsaturated ketones from saturated ketones are (a) halogenation followed by dehydrohalogenation, (b) utilising sulphur or selenium derivatives, (c) DDQ and (d) utilizing palladium(II) complexes.
Furthermore, it has also been found that by following the prior art methods mentioned above, estetrol of high purity was obtained only in low yield when using an acetyl group as a protecting group for the 3-hydroxy group of estra-1,3,5(10),15-tetraen-3-ol-17-one, in particular because its sensitivity to hydrolysis and solvolysis. In particular, the lability of the acetyl group lead not only to an increased formation of byproducts during the reactions, but also during chromatography and crystallisation for purification of intermediate products when protic solvents such as methanol were used. Therefore, it is difficult to isolate purified estetrol and intermediates thereof in good yield.
Additionally, the reduction of the carbonyl group at C17 with LiAlH4 proceeds with a low selectivity since various amounts of β-estradiol (estra-1,3,5(10)-trien-3,17β-diol) are obtained as well. Obviously, the formation of such a by-product reduces the yield as well as the purity of the desired product which requires additional purification steps.
The prior art methods also employ stoichiometric amounts of OsO4 in the oxidation step that is known to be a toxic and expensive compound. Consequently, the use of such a reagent is undesired in view of safety and operational costs.
Accordingly, it is an object of the present invention to provide a synthesis route for estetrol whereby high yields and high purities of estetrol are obtained.
Still accordingly, there is a need for a synthesis of estetrol wherein the production of by-products is limited. i.e. preferably less than its detection level.
It is a preferred object of the invention to provide a synthesis of estetrol wherein good yield and good purity of estetrol are provided.
By a good yield, it is meant a yield of at least 10%, preferably higher than 10%, more preferably of at least 12.5%, starting from estrone (100%).
By a good purity, it is meant a purity of at least 97%, preferably of at least 98%, more preferably of at least 99%. Preferably, single impurities are not allowed to exceed 1%. Also preferred is that β-estradiol is not allowed to exceed the detection level.
For the purpose of the present invention, determination of purity is made by HPLC-MS. The following conditions are used:
HPLC-MS is performed using a Hewlett Packard 1100 series:
Column: Discovery C18 (150×4.6 mm) Supelco
Mobile phase: Solution A:Solution B=70:30 (5 min)→(10 min)→10:90 (5 min)
Flow: 1 mL/min
UV: 280 nm
Temp: 22° C.
MS: API-ES negative
Solution A: 9.65 g NH4OAc, 2250 mL H2O, 150 mL MeOH, 100 mL CH3CN
Solution B: 9.65 g NH4OAc, 250 mL H2O, 1350 mL MeOH, 900 mL CH3CN
It has now been found that protecting the 3-OH group of estra-1,3,5(10),15-tetraen-3-of-17-one by an C1-C5 alkyl group, preferably a methyl group, or a C7-C12 benzylic group, preferably a benzyl group, fulfils such a need. Indeed, it has been found that the use of a more stable protective group such as a C1-C5 alkyl group, preferably a methyl group, or a C7-C12 benzylic group, preferably a benzyl group, on the 3-OH group is not cleaved at an undesired stage of the synthesis. Therefore the formation of by-products is limited and the purification of intermediates is more practical.
In this patent application the term “alkyl” includes linear, branched and cyclic alkyl groups such as methyl, ethyl, n-propyl, i-propyl, c-propyl, n-butyl, s-butyl, t-butyl, c-butyl, n-pentyl, s-pentyl, t-pentyl, c-pentyl and methylcyclobutyl. Additionally, the C7-C12 benzylic group has to be understood as a benzyl group that may be substituted with one or more substituents at the ortho, meta and/or para position of the aromatic nucleus, wherein the substituents are aliphatic groups, optionally substituted by one or more heteroatoms and/or halogen atoms that do not adversely interfere with the synthetic process. As is obvious to a skilled person in the art, the alkyl and benzylic groups are intended as a protecting group and these groups must therefore be relatively easy to add and relatively easy to remove under such conditions that do not have an adverse effect on the molecular structure of the estrone derived steroid molecules.
Because of the selected protecting groups which are used and the yield and purity obtained, it appeared that the synthesis disclosed in this patent application can be suitably transposed to an industrial scale. This represents a particular advantage in comparison to the current lab-scale syntheses which have been disclosed in the prior art and which hamper from several disadvantages as disclosed above. Indeed, a problem with industrial syntheses are the quantities of chemicals as well as the toxicity and hazardous properties thereof which are involved, thus making the prior art lab-scale methods not transposable to an industrial scale. The reason behind such impossible replication is that usually the known method either does not provide a sufficient yield, i.e. at least 10% to be considered economically feasible from an industrial point of view and/or produce by-product(s) which necessitates at least a subsequent purification step, thus raising the cost of the method.
Accordingly, it is also another preferred object of the invention to provide a method which is suitable for use in industry.